Treatment of human immunodeficiency virus/acquired immunodeficiency syndrome

ABSTRACT

Methods of treating a patient with human immunodeficiency virus are disclosed. The method includes a providing intradermal and intravenous doses of a aTh1 composition that can increase the CD4+ cells in a patient that are resistant to HIV. The description includes a method for viral load reduction and a viral purge method. The regimen leads to a spike in the viral load and a then a return to baseline or lower levels of the virus and can lead to reduction and/or elimination of the latent viral reservoirs. Kits configured to provide intradermal doses and intravenous doses according to the regimen are also included.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional of and claims the benefit ofU.S. patent application Ser. No. 14/592,701, filed Jan. 8, 2015, whichis based on and claims the benefit of U.S. provisional patentapplication Ser. No. 61/924,936, filed Jan. 8, 2014, the contents ofwhich is hereby incorporated by reference in their entirety.

FIELD

This disclosure relates to treatment of anti-retroviral therapy and moreparticularly relates to immunotherapy treatment of HIV/AIDS.

BACKGROUND

AIDS was first reported in the United States in 1981 and has sincebecome a major worldwide pandemic. AIDS is caused by the humanimmunodeficiency virus, or HIV. Today more than 30 million people livingthroughout the world are infected by the virus (Cohen, Hellmann et al.2008). HIV progressively destroys the body's ability to fight infectionsand other diseases by killing or damaging cells of the body's immunesystem, specifically eliminating immune cells that express the CD4molecule, such as CD4+ helper T-lymphocytes (leading to an invertedCD4/CD8 T-cells ratio) and cells of the monocyte/macrophage lineage(Fauci 1996).

CD4 T-cells mature into two polarized functional types, called Th1 andTh2 (Mosmann and Coffman 1989; Mosmann and Sad 1996). Th1 CD4+ cells areresponsible for mediating cellular immunity and Th2 CD4+ cells areresponsible for mediating humoral immunity (D'Elios and Del Prete 1998).HIV infection causes a gradual loss of the Th1 subset resulting in aninverted Th1/Th2 ratio (Becker 2004) and loss of cellular immunity. Theloss of Th1 immunity and switch to Th2-dominated immunity in HIVpatients has been correlated with profound immunosuppression and theprogression from HIV positive status to AIDS (Klein, Dobmeyer et al.1997). One of the leading causes of death of patients with AIDS isopportunistic infections due to the suppression of the cellular immunesystem (Baker and Leigh 1991).

HIV has multiple strategies for immune evasion. These strategies includemutational escape, latency, masking of antibody-binding sites on theviral envelope, down-modulation of the class I major histocompatibilitycomplex (MHC-I), up-regulation of the Fas ligand on the surface ofinfected cells (Piguet and Trono 2001) and inducing the production ofIL-10 (Leghmari, Bennasser et al. 2008; Brockman, Kwon et al. 2009). Inaddition, Some viral genes such as vif, vpr, vpu, and nef genestranslate proteins that act to suppress anti-viral immune responses(Kirchhoff 2010). These viral escape mechanisms make the virus elusivefor control using immunological methods (Migueles, Tilton et al. 2006;Bansal, Yue et al. 2007; Feinberg and Ahmed 2012; Teshome and Assefa2014).

HIV virology has been intensively studied and the viral structure andlife cycle of HIV has been described (Pomerantz 2002; Sierra, Kupfer etal. 2005; Li and Craigie 2006; Cohen 2008; Scherer, Douek et al. 2008;Fanales-Belasio, Raimondo et al. 2010). A single HIV particle is calleda virion. The virion is shaped like a spiked sphere. The central core ofthe sphere is called the capsid. The capsid contains two single strandsof HIV RNA called viral RNA. When viral RNA is detected in the serum,the quantity of viral RNA is called the viral load. The viral RNA codesfor three enzymes important to the virus's life cycle called reversetranscriptase, integrase, and protease. These enzymes are foreign tohuman immune system and are capable of being recognized by CD8+ CTLkiller cells (Haas, Samri et al. 1998). In this manner, cells thatexpress these viral enzymes are targets for immune elimination. However,the viral RNA also contains instructions for production of viralaccessory proteins that serve to assist the virus to evade immuneelimination (Seelamgari, Maddukuri et al. 2004; Malim and Emerman 2008).

Surrounding the core is a protective lipid (fat) bilayer which forms ashell around the capsid (Frankel 1996; Bradbury 2013). This shell iscalled the viral envelope. Embedded within the viral envelope is a HIVprotein called env. The env protein is made up of two glycoproteins,gp120 and gp41, that protrude from the virion forming the spikes. Thecap of the spike is gp120 and the stem is gp41. For HIV to enter a hostcell, it must first use gp120 to attach to a CD4 receptor (Pancera,Majeed et al. 2010; Guttman and Lee 2013).

After gp120 successfully attaches to a CD4 cell, the molecule can changeshape to avoid recognition by neutralizing antibodies, a process knownas conformational masking (Kwong, Doyle et al. 2002). The conformationalchange in gp120 allows it to bind to a second receptor on the CD4 cellsurface called a chemokine receptor.

The chemokine receptor on the CD4 cells surface used as a co-receptorfor the HIV virion is either CCR5 or CXCR4 (Moore, Trkola et al. 1997).The viral preference for using one chemokine co-receptor versus anotheris called ‘viral tropism’. Chemokine receptor 5 (CCR5), is used bymacrophage-tropic (M-tropic) HIV to bind to a cell (Cohen, Kinter et al.1997). About 90% of all HIV infections involve the M-tropic HIV strain.CXCR4, also called fusin, is a chemokine receptor used by T-tropic HIV(ones that preferentially infect CD4 T-cells) to attach to the host cell(Hoxie, LaBranche et al. 1998). Another co-receptor called DC-SIGN isexpressed on dendritic cells and also binds gp120 in order to facilitateviral infection of these important cells involved in cellular immunity(Cunningham, Harman et al. 2007). Viral infected macrophages caninteract with CD4 T-cells and pass the virus through cell-to-cellcontract (Martin and Sattentau 2009; Poli 2013). In addition, HIV caninduce T-cells to form syncytium to facilitate cell-to-cell viraltransfer (Emilie, Maillot et al. 1990; Kozal, Ramachandran et al. 1994;Margolis, Glushakova et al. 1995).

Transmission of HIV results in the establishment of a new infection,starting from even a single virion particle. HIV virons are replicatedwithin host infected cells and released into the plasma which causesviremia and persistent infection of immune cells in all of the lymphoidtissues in the body. HIV preferentially infects T cells with high levelsof CD4 surface expression and those subsets of T cells that co-expressCCR5. The subset of memory T-cells are a preferred target (Helbert,Walter et al. 1997), particularly HIV-specific memory T cells (Douek,Brenchley et al. 2002) and Th2/Th0 cells (Maggi, Mazzetti et al. 1994).

With the onset of immunodeficiency, the virus evolves to infect new celltypes. This correlates with a tropism change involves switching frompreference for CCR5 co-receptor to the alternative CXCR4 co-receptors.This switch corresponds with an expansion of infected cells to includenaive CD4+ T cells in addition to the preferred memory cells. Similarly,the virus evolves the ability to enter cells with low levels of CD4 onthe surface and this potentiates the ability to infectmonocyte/macrophages. Naive cells are found almost exclusively in thesecondary lymphoid organs, while memory cells and macrophages have amuch wider tissue distribution, including the brain, tissue and organsystems. Infection of naive cells and macrophages establishes pools ofviral infected cells throughout the body and in locations that aredifficult to target with drugs or immunotherapy.

M-tropic and T-tropic strains of HIV can also coexist in the body,further complicating the ability to target elimination of the virus. Atsome point in infection, gp120 is able to attach to either CCR5 orCXCR4. A HIV virion with this property is called a dual tropic virus orR5X4 HIV (Toma, Whitcomb et al. 2010; Loftin, Kienzle et al. 2011;Svicher, Balestra et al. 2011). HIV that can utilize the CXCR4 receptoron both macrophages and T-cells is also termed dual-tropic X4 HIV(Huang, Eshleman et al. 2009; Gouwy, Struyf et al. 2011; Xiang, Pachecoet al. 2013). Mixed tropism results when an individual has two viruspopulations; one using CCR5 and the other CXCR4 to bind to the CD4T-cell. Since the virological behavior of T-tropic and M-tropic virusesvary, mixed tropism creates a difficult problem for drug design.

Once the HIV envelope has attached to the CD4 molecule and is bound to aco-receptor, the HIV envelope utilizes a structural change in the gp41envelope protein to fuse with the cell membrane and evade neutralizingantibodies (Chen, Kwon et al. 2009). The HIV virion is then able topenetrate the target cell membrane.

Once within a host cell, the viral enzyme reverse transcriptase convertsthe viral RNA to viral DNA. Reverse transcriptase inhibitors aredeveloped as an anti-HIV therapy (Nurutdinova and Overton 2009; Chowers,Gottesman et al. 2010; Zhan and Liu 2011). Once the viral RNA istranscribed to DNA, the DNA is then able to enter the nucleus of thehost cell. Using another viral enzyme called integrase, the viral DNA isable to integrate into the host cell's chromosomal DNA. Integraseinhibition is another target of anti-viral drug development (Geretti,Armenia et al. 2012; Okello, Nishonov et al. 2013). The integrated viralDNA is called provirus and is replicated along with the host chromosomewhen the host cell divides. The integration of provirus into the hostDNA provides the latency that enables the virus to effectively evadehost immune responses.

When the host cell is activated to divide, production of viral proteinsand viral RNA takes place as the provirus is transcribed along with thehost DNA. Viral proteins are then assembled using the host cell'sprotein-making machinery. The virus's protease enzyme allows for theprocessing of newly translated viral polypeptides into the proteinswhich constitute the virus. These various proteins are then ultimatelyassembled into viral particles. Protease inhibitors are another class ofanti-viral drugs for treatment of HIV infection (Wattanutchariya,Sirisanthana et al. 2013). The assembled virus uses the nuclear capsidprotein called gag to interact with host protein machinery to cause thebudding of the virus and release of whole virus from the host cell(Dussupt, Javid et al. 2009). Alternatively, the budding HIV cantransfer directly from cell-to-cell interaction (Fais, Capobianchi etal. 1995). Many viral particles can bud from of a single cell over thecourse of time, eventually lysing the cell membrane killing the cell.

Cells actively producing virus are vulnerable to attack by CD8 cells(cytotoxic T-lymphocytes or CTLs). CTL cells require help from Th1 CD4cells to kill cells that are producing virus (Wodarz 2001). In HIVinfection, the viral load can be kept in a steady state with the rate ofimmune-mediated destruction of viral producing cells balanced with therate of release of viral particles from infected cells. In this steadystate, the viral load is maintained at a set point level (KorthalsAltes, Ribeiro et al. 2003; Kaul, MacDonald et al. 2010). When CD4counts drop sufficient to lose this helper function for CTL, the setpoint control is lost and the viral load climbs. Eventually this leadsto a fall in CD4 counts, loss of cellular immunity and eventuallyleading to AIDS. An HIV infection can be in such a steady state foreight to ten years before the clinical syndrome of AIDS occurs(Jurriaans and Goudsmit 1996; Callaway and Perelson 2002; Maenetje, Riouet al. 2010).

The most obvious laboratory observation in HIV infection is a decline inthe number of CD4+ T− cells found in the blood and a decline in theCD4/CD8 ratio. Increase in viral load (viral RNA) can be detected bysensitive PCR tests.

Highly active antiretroviral therapy (HAART) for the chronic suppressionof HIV replication has been the major accomplishment in HIV/AIDSmedicine. HAART cocktails contain drugs with different mechanisms ofaction designed to block the natural virus life cycle at differentpoints. For example, HAART can contain reverse transcriptase, integrase,protease and binding (Carter 2003; Laurence 2004; 2007) inhibitors. Manypatients are now in their second decade of treatment, with levels ofplasma HIV RNA (viral load) below the limits of detection of clinicalassays (e.g., <50 copies/ml). New HAART drugs are being developed tointerfere with the viral life cycle. For example, since CCR5 has beenidentified as a major HIV co-receptor this has lead to the developmentof drugs that target the virus-CCR5 interaction, including thefirst-in-class approved drug, Maraviroc (Rusconi, Vitiello et al. 2013).

Since HAART is not able to completely eliminate the virus, life-longantiviral therapy is needed to control HIV infection. Such therapy isexpensive and prone to drug resistance, cumulative side effects andunknown effects of long-term treatment. HAART has several long-term sideeffects including kidney, liver, and pancreatic problems; and changes infat metabolism, which result in elevated cholesterol and triglyceridelevels and an increased risk for strokes and heart attacks (Carter 2003;Laurence 2004; 2007). In addition, some viruses have evolved resistanceto HAART (Fumero and Podzamczer 2003; Tebit, Sangare et al. 2008;Loulergue, Delaugerre et al. 2011).

HIV infection persists in spite of efficacious HAART therapies asevidenced by rapid rebound of viremia upon cessation of HAART therapymost often within 3-10 days (Neumann, Tubiana et al. 1999; Van Gulck,Heyndrickx et al. 2011). This phenomenon is thought to be due to theearly establishment of a stable reservoir of latently infected cellswith integrated viral DNA that seeds the production of virions afterHAART cessation.

The goal of HAART therapy in HIV-infected patients is to reduce plasmaHIV viral load (HIV RNA) to undetectable levels and to increase the CD4cell count. Achievement of this goal reduces the rate of diseaseprogression and death. However, some patients experience isolatedepisodes of transiently detectable HIV RNA or viral rebound (Staszewski,Miller et al. 1998; Butler, Gavin et al. 2014). The causes of viralrebound are still unclear. Rates of viral rebound of 25-53% have beenreported among patients on HAART who have achieved undetectable HIV RNA.Viral rebound that then persists as a low level viremia (set pointlevel) may lead to genetic mutations in the virus leading to drugresistance.

Patients with persistent low-level viremia have a higher rate ofvirological failure. Persistent low-level viremia is defined as plasmaHIV RNA levels in the range of 51-1000 copies/mL for at least 3 monthsand on at least two consecutive clinic visits. Virological failure isdefined as two consecutive plasma HIV RNA levels >1000 copies/mL.

After HAART initiation, most patients experience improved immunefunction and maintain viral suppression; however, there remains a subsetof patients who have suboptimal immunologic responses—defined as thefailure to achieve and maintain an adequate CD4 response despite use ofHAART therapy. Patients with inadequate CD4 counts on HAART therapy aresaid to have immunological failure. Adequate CD4 counts are generallydefined as >500 cells/mm³ over a specific period of time (e.g., 4 to 7years). Immunological failure increases the risk of AIDS- andnon-AIDS-related morbidity and mortality. For example, a low CD4 countof <500 is associated with an increased risk in cardiovascular, hepatic,and renal disease and cancer.

Cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell responses areimportant to the initial decrease in HIV viral load seen in the firstseveral months after acute infection (Borrow, Lewicki et al. 1997; Fan,Huang et al. 1997; Smalls-Mantey, Connors et al. 2013). These beneficialcellular immune responses diminish with disease progression and cannotbe recovered with antiretroviral therapy alone. CTL responses generallyrequire CD4 cell help to be effective (Wodarz 2001).

Recent studies suggest a therapeutic vaccine may help to restorecellular immunity and CTL and NK responses to the virus. Therapeutic HIVvaccines are designed to control HIV infection by boosting the body'snatural immune response. HIV-specific T-cell-based vaccines have beenextensively studied in both prevention and therapeutic settings, withmost studies failing to show benefit, and some suggesting harm (Papagno,Alter et al. 2011). There are currently no FDA-approved therapeutic HIVvaccines.

So far it has been impossible to cure HIV despite long-term viralsuppression on HAART. The rapid rebound despite powerful viralsuppression and blockage of viral entry is thought to be due to thereservoirs of latently infected cells unaffected by viral suppressionand unable to be targeted for immune elimination, also the continuoussub-clinical viral production from some cells in lymph nodes and tissuesand the ability of the virus to spread through cell-to-cell contact asan alternative to entry pathway all serve to maintain viral persistence.

While there are descriptions of some patients that can remain withundetectable virus without HAART, these so called “secondarycontrollers” are infected with less infectious types of HIV (Lobritz,Lassen et al. 2011; Van Gulck, Bracke et al. 2012). For the majority ofpatients, HAART is a lifetime requirement for disease control.

The only report of long-term viral suppression after cessation of HAARTtherapy is the so called “Berlin Patient”. The Berlin Patient receivedan allogeneic stem cell transplant for treatment of his leukemia. Thedonor had a special genetic characteristic (two copies of the recessiveCCR5Δ32 allele) which results in the inability to express the CCR5receptor on the surface of CD4 cells. Thus the donor cells for thetransplant were resistant to viral entry. After transplant, the patientwas able to stop all HAART anti-retroviral therapy and remained withundetectable viral load for 3½ years after the transplant (Hutter, Nowaket al. 2009).

It is possible that innate or acquired immunity delivered by the donorimmune system may have contributed to the elimination of cells withactive HIV replication. The patient experienced graft versus hostdisease (GVHD), and it is possible that an allogeneic immune responsedirected against host lymphocytes had a purging effect on the latent HIVreservoir in lymphocytes.

Allogeneic stem cell transplant is a highly toxic procedure with hightreatment related mortality and morbidity. The high toxicity is relatedto the need for chemotherapy conditioning regimes and to the oftenlethal GVHD side-effect. The toxicity of GVHD limits the clinical use ofallogeneic transplant procedures to terminally-ill patients withoutother treatment options. However, in HIV+ patients that are stable onHAART medication, it is not clinically feasible to treat with allogeneicstem cell transplant.

Further, allogeneic transplant requires HLA tissue matched donors. Only⅓ of individuals have a related HLA-matched donor and fewer are able tofind an unrelated HLA matched donor. Moreover, even if a matched donorcan be identified, the donor must be homozygous for the CCR5Δ32mutation, which is an extremely rare genetic phenotype (Jiang, Wang etal. 1999; Williamson, Loubser et al. 2000). Thus the lack of suitabledonors and the toxicity of allogeneic transplant procedures makes itimpossible to translate data from the Berlin patient to benefit themajority of HIV infected patients.

Accordingly, additional non-toxic therapies are needed in order toexploit the mechanisms that enabled the Berlin Patient to enjoylong-term HAART cessation. In addition, treatment options for virologicfailure and immunological failure while on HAART treatment are urgentlyneeded.

SUMMARY

The present description relates to an immunotherapy drug and atherapeutic vaccine composition and methods of use for treating patientswith HIV infection that experience virological and/or immunologicalfailure while on HAART medication. In addition, the present descriptiondescribes a method for purging latent viral pools in HIV patients tolevels sufficient to enable extended holiday from the requirement fordaily HAART medication.

The compositions of the present description include a combination ofliving cells, or components thereof, containing at least one highlyimmunogenic antigen, a molecule that binds surface CD40 receptors todeliver cellular activation signals and one or more inflammatory type 1cytokines and/or chemokines delivered together or separately in time(hereinafter referred to as “aTh1”). The aTh1 composition may alsoinclude at least one anti-retroviral drug. The aTh1 composition thatincludes anti-retroviral drug may be referred to herein as the AVIcomposition. The aTh1 composition and anti-viral drugs can be deliveredby different routes but the effects of both must be concurrent. Thecomponents of the aTh1 composition can be combined in a solution orattached to a surface, such as a biodegradable support, foradministration. An exemplary aTh1 composition is known as “AlloStim™,”and can be obtained from Immunovative Therapies, Ltd.

The present description includes a method for enhancing CD4+ T-cells inthe patient. This CD4 Enhancement Method” includes using the aTh1composition for increasing the titer of circulating CD4+ Th1 cells inHIV patients, including CD4+ cells that are resistant to HIV infectiondue to having a memory phenotype and down-regulation of surface CCR5expression or blocking of CCR5 due to production of chemokine agonistsor both. This method can be used concurrently with HAART in HIV patientsexperiencing immunological failure.

The present description also includes a therapeutic vaccine method. Themethod includes using aTh1 composition as an adjuvant together with asource of HIV antigen forming a therapeutic vaccine that results inincreased titer of HIV-specific T-cells and immune control of the virus.This method can be used as a therapeutic vaccine in HIV patients,including patients on HAART medication experiencing virological failure.

The present description also includes a viral purge method. This methodincludes using the AVI composition for activating cells latentlyinfected with HIV genetic material so that they produce viral particlesand thus become targets for immune-mediated elimination. The anti-viralmedication in the AVI composition prevents the awakened latent viruspool to overwhelm and destroy the remaining CD4 cells. This method canbe used to decrease or eliminate the latent viral pool. Purging of thelatent viral pool is a required step for an eventual cure.

In another aspect of the description, a HIV treatment method isdescribed which combines the CD4 Enhancement method with the Viral Purgemethod (“HAART Holiday Method”). The HAART Holiday Method can also becombined with the Therapeutic Vaccine Method. The HAART Holiday Methodprovides HIV patients with an extended holiday from the dailyrequirement for HAART medications. Such a holiday is preferably longerthan 30 days, more preferably for at least 90 days and most preferablyfor more than a year.

In one aspect, the present description includes a method of treating apatient with HIV. The method includes increasing the titer ofcirculating CD4+ Th1 memory cells that are resistant to HIV infection byadministering at least one intradermal dose of aTh1 composition to thepatient wherein the patient is infected with HIV. The method furtherincludes expanding and activating the CD4+ Th1 memory cells in thepatient by administering at least one intravenous dose of the aTh1composition. The method may also includes increasing the titer byadministering at least two intradermal doses of aTh1 composition,wherein both of the doses are at the same location and the intervalbetween the intradermal doses is between about 3 days and about a week.The method may also include an additional two intradermal doses of theaTh1 composition at a location different than the location of the firsttwo intradermal doses. The method may also include wherein the patientis concurrently treated with highly active antiretroviral therapy(HAART).

In another aspect, the present description also includes a method ofreducing the viral load in a patient with HIV. The method includesadministering at least one dose of a aTh1 composition and at least onedose of one or more HIV-antigens, wherein the titer of circulating CD4+Th1 memory cells that are resistant to HIV infection are increased inthe patient and the viral load is decreased in the patient. The methodmay include wherein the aTh1 composition and the HIV-antigens areadministered separately aTh1 composition and the one or moreHIV-antigens are administered intradermally.

In yet another aspect, the present description also includes a method ofreducing or eliminating HIV-virus from a patient. The method includesescalating intravenous doses of a aTh1 composition to a patient whereinthe patient is concurrently treated with HAART. The method may alsoinclude a step wherein the HAART is halted and the patient is monitoredfor CD4+ cells and the viral load and wherein HAART is reinstated if aviral spike is detected in the patient.

In a further aspect, the present description includes a kit comprisingcomponents of a therapeutic HIV vaccine wherein the kit comprisesintradermal doses of a aTh1 composition, intravenous doses of a aTh1composition and one or more HIV-antigens. The kit may further includecomponents of HAART.

In yet a further aspect, the present description includes a compositionincluding a aTh1 composition comprising an alloantigen, a molecule thatinteracts with CD40 surface receptor and Type I cytokines and at leastone or more HIV-antigens.

In another further aspect, the present description includes a an AVIcomposition that includes a aTh1 composition comprising an alloantigen,a molecule that interacts with CD40 surface receptor and Type Icytokines and at least one or more anti-retroviral drugs. Thecomposition may also include one or more HIV-antigens.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This present description includes compositions for patients infectedwith retroviruses, especially Human Immunodeficiency Virus (HIV). Thecomposition can include the aTh1 composition for eliciting animmunological response by the patient. The present description alsoincludes an anti-viral immunotherapy drug composition (aTh1+anti-viralmedication) and methods for using the anti-viral composition fortreatment of patients infected with HIV. Methods are described whereinthe compositions can be used to: (1) treat immunological failure byincreasing the CD4 count (CD4 Enhancement Method); (2) treat virologicalfailure by restoring immune control of viral load (Therapeutic VaccineMethod); and (3) purge virus from the latent viral pool (Viral PurgeMethod). The combination of all these methods or the combination of theCD4 Enhancement Method and the Viral Purge Method can eliminate the needfor daily HAART medication for an extended period of time (HAART HolidayMethod).

The anti-viral immunotherapy composition comprising aTh1 and anti-viralmedication may be referred to herein as AVI composition.

Patients infected with HIV can be treated with the compositions andmethods described herein. The patients may be treated while experiencingimmunological or viral failure while on HAART. The patient may betreated with or without simultaneous HAART medication. A biomarker forsuccessful treatment by the composition and methods described herein canbe characterized by enhanced serum levels of IL-12 in plasma of the HIVpatients. IL-12 can enhance HIV-specific cellular immunity. The methodsof the description generally can cause the appearance of IL-12 in theserum by at least 120 days of administering the aTh1 composition,preferably by 90 days, more preferably by 30 days and even morepreferably by 7 days. IL-12 can serve as an early biomarker indicatingsuccess of the methods in creating anti-HIV immunity.

The aTh1 composition can include i) living cells, or components thereof,containing at least one highly immunogenic antigen, ii) a molecule thatdelivers a signal through binding to surface CD40 receptor and iii) oneor more inflammatory type 1 cytokines and/or chemokines. All of thesecomponents of the aTh1 composition can be delivered together orindividually at the same time or separately in time.

The highly immunogenic antigen component of the aTh1 composition can benatural, synthetic or recombinant proteins or peptides that have someforeign component that can make them recognizable to the human immunesystem. The immunogenic antigens can be, for example, allogeneic orxenogenic protein antigens. Self proteins that are altered to berecognized as foreign are also within the scope of the description. Thealteration of the self-protein can be by recombinant or chemical meansor by mixing the self-protein with an adjuvant. In a preferredembodiment, the highly immunogenic antigen is part of a living cell,preferably an allogeneic living cell, more preferably a livingallogeneic immune cell, most preferably an allogeneic living Th1 immunecell. Alloantigens are a preferred highly immunogenic antigen includedin the aTh1 composition.

The highly immunogenic antigen(s) of the composition can be capable ofbeing processed by professional antigen presenting cells (APC) forpresentation on MHCI and/or MHCII molecules. Examples of highlyimmunogenic antigens may include also KLH, viral proteins, bacterialprotein, yeast proteins, fungal proteins or combinations thereof.

Examples of adjuvants that can increase the immunogenicity of a protein,such as a self protein, include agents which cause immature dendriticcells to mature to IL-12+ DC1 cells. Examples include adjuvant dangersignals such as LPS, BCG and Toll-Like receptor agonists (e.g., TLR4 andTLR7). All highly immunogenic peptides and proteins are within the scopeof this description.

The aTh1 composition can also include type I cytokines and/orchemokines. Preferred Type 1 cytokines for the aTh1 composition caninclude interferon-gamma, IL-2, TNF-alpha, TNF-beta, GM-CSF, IL-1, IL-7,IL-15, IL-23 and IL-12 individually or in combinations thereof.Preferred chemokines for the aTh1 composition can include RANTES,MIP-1alpha, MIP-1beta and MCP-1 individually or in combinations thereof.These type I cytokines can either be part of the aTh1 composition or canbe induced in the patient by the aTh1 composition.

The aTh1 composition can also include a molecule that delivers a signalthrough surface CD40 receptor. One preferred molecule in the aTh1composition that delivers a signal through CD40 is immobilized CD40L(CD154). CD40L (also known as CD154) is a member of the TNF superfamily.CD40L can act as a co-stimulatory molecule that interacts with CD40expressed on dendritic cells (DC) to support their maturation to aIL-12+ phenotype. CD40L is preferably immobilized by expression on acell surface so that it provides a positive signal through CD40.Alternatively, an agonist to CD40 can be used to deliver a CD40 signal,such as a fusion protein or an anti-CD40 antibody. The components of theaTh1 composition can be delivered together or separately and in varioussequences and at various points in time and are within the scope of thisdescription.

In preferred embodiments, the aTh1 composition can include activatedallogeneic CD4+ T-cells, and in more preferred embodiments, allogeneicactivated memory CD4+ T-cells with high surface expression of CD40L andwhich produce interferon-gamma, are used.

IL-12 production and CD40L expression in HIV-infected (HIV+) individualscan be severely impaired. CD40-CD40L interactions are the majormechanism involved in the T cell-dependent activation ofantigen-presenting cells (APC), such as DC, to produce IL-12. WhileCD40, the counter-receptor for CD40L, is expressed on monocytes fromHIV+ individuals, IL-12 production can still be suppressed. Theappearance of IL-12 in the plasma after administration of the aTh1composition can indicate successful initiation of the immunologicalmechanism of the methods.

Different forms of CD40L can also signal through CD40. For example,soluble trimeric CD40L agonist protein (CD40LT), soluble CD40L and CD40Linserted into HIV virus can also provide the same signal and the sameeffect. All forms of CD40 agonist are within the scope of thisdescription.

In some preferred embodiments, the aTh1 composition can be AlloStim™.AlloStim™ are bioengineered CD4 immune cells derived from the blood ofnormal donors. AlloStim™ has an activated Th1 memory phenotype: CD4+,CD45RO+, CD62L^(lo), CD40L^(hi), CD25+, interferon-gamma+ and IL-4−.AlloStim™ can be maintained in an activated state by continuousattachment to CD3/CD28-monoclonal antibody-coated microparticles. Thekey effector molecules of AlloStim™ are the high surface expression ofCD40L and the production of high amounts of inflammatory cytokines, suchas interferon-gamma, tumor necrosis factor-alpha andgranulocyte-macrophage colony stimulating factor (GM-CSF). AlloStim™ andmethods of making AlloStim™ are described, for example, in U.S. Pat.Nos. 7,435,592, 7,678,572 and 7,402,431, all incorporated herein byreference. Other allogeneic or xenogeneic immune cells can also be usedas components in the aTh1 composition. Some of the methods of thepresent disclosure are described with reference to AlloStim™ but this isnot meant to limit the methods to the use of AlloStim™ only and othercompositions may be used in the described methods.

The aTh1 compositions described herein may also include anti-viral oranti-retroviral medication (AVI composition). Compositions such asAlloStim™ which contain the necessary components of the aTh1 compositionhave been previously disclosed. In some embodiments, the use ofAlloStim™ alone or aTh1 composition may not be sufficient for treatingHIV infection. The AVI composition includes anti-retroviral medicationtogether with the aTh1 composition.

While the aTh1 composition can be beneficial for cancer treatment, thiscomposition may be detrimental to an HIV patient. This is due to theunique nature of the HIV life cycle. For example, when AlloStim™ is usedas the aTh1 composition, intradermal injections of AlloStim™ canincrease the titer of memory CD4+ cells specific for alloantigens. InHIV infection, this increase in CD4+ memory cells alone would onlyincrease the number of CD4+ targets for the virus to infect. If thepatient was not viral suppressed to have viral load below the limit ofdetection, circulating virions would infect the newly formed CD4 cellsincreasing the pool of latent virus. Thus, intradermal AlloStim™injections alone will lead to increase latent viral pool. The feature ofthe present method that can protect these newly formed CD4+ cells fromviral entry is a step that can activate these cells using intravenousinfusions. Activated memory cells can be resistant to viral entry due toup-regulation of CCR5 agonist cytokines and down-regulation of the CCR5receptor. However, the mass activation of memory cells can awaken theviral production of latently infected cells. Intravenous infusions ofAlloStim™ can cause activation of T-cells and monocytes that can causeany latently infected cells to begin viral production. This can resultin an increase in plasma viral load and can eventually lead to adecrease in CD4+ cell counts. Further, the activation of latent viralpools after intravenous AlloStim™ infusion and the subsequent increasein viral replication may lead to increased risks in the development ofviral escape mutants that become resistant to HAART drug cocktails.Carefully sequenced administration by dose and route can be required inorder to treat HIV using the aTh1 compositions such as AlloStim™, whencombined with anti-retroviral medications such as used in HAART. Thiscan slow the production of virus and can allow for establishment ofimmune control of the virus. Frequent monitoring of CD4 counts and HIVRNA viral load can be performed to assure the proper balance ismaintained. The latent viral load can be monitored by monitoring bothcellular and plasma viral DNA levels.

AlloStim™ or other aTh1 compositions can be used initially together withviral suppressing drugs to slow down the spread of virus to healthycells and prevent viral mutation.

A variety of anti-retroviral drugs or medications can be included in theAVI composition. The AVI composition can include, for example, one ormore drugs from any of the classes of antiretroviral drugs. Theanti-retroviral drugs, for example, can include drugs from the followingclasses. Drugs from other classes are also within the scope of thisdescription.

Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs):

Sometimes called “nukes.” These anti-HIV drugs can work to block HIV'sability to use reverse transcriptase to correctly change viral RNA intoDNA. Host cells can use DNA to produce the proteins that the virus needsto make copies of itself.

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs):

These are called “non-nukes.” They can work in a very similar way to“nukes.” Non-nukes also can block the enzyme, reverse transcriptase, andcan also prevent HIV from making copies of its own DNA. But unlike thenukes (which work on the genetic material), non-nukes can act directlyon the enzyme itself to prevent it from functioning correctly.

Protease Inhibitors (PIs):

When HIV replicates inside cells, it can create long strands of its ownRNA genetic material. These long strands have to be cut into shorterstrands in order for HIV to create more copies of itself. The enzymethat acts to cut up these long strands is called protease. Proteaseinhibitors can block this enzyme and prevent those long strands ofgenetic material from being cut up into functional pieces.

Entry/Fusion Inhibitors:

These medications can work to block the virus from entering cells. HIVattaches and bonds to CD4 cells through receptor sites. Receptor sitesare found on both HIV and CD4 cells (they are found on other types ofcells too). Fusion inhibitors can target those sites on either HIV orCD4 cells and can prevent HIV from “docking” into healthy cells. CCR5 isan example of a receptor site for HIV.

Examples of anti-HIV drugs that can be included in the AVI compositionmay include the following multi-classcombinations: Atripla(efavirenz+tenofovir DF+emtricitabine); Complera (Eviplera,rilpivirine+tenofovir DF+emtricitabine); Stribild (formerly Quad)(elvitegravir+cobicistat+tenofovir DF+emtricitabine); Triumeq (formerlyTrii) (dolutegravir+abacavir+lamivudine).

Examples of anti-HIV drugs include the following NNRTs: Edurant(rilpivirine, RPV, TMC-278); Intelence (etravirine, ETR, TMC-125);Rescriptor (delavirdine, DLV); Sustiva (Stocrin, efavirenz, EFV);Viramune and Viramune XR (nevirapine, NVP); Lersivirine (UK-453061).

Examples of anti-HIV drugs include the following NRTIs: Combivir(zidovudine+lamivudine, AZT+3TC); Emtriva (emtricitabine, FTC); Epivir(lamivudine, 3TC); Epzicom (Kivexa, abacavir+lamivudine, ABC+3TC);Retrovir (zidovudine, AZT, ZDV); Trizivir(abacavir+zidovudine+lamivudine, ABC+AZT+3TC); Truvada (tenofovirDF+emtricitabine, TDF+FTC); Videx EC and Videx (didanosine, ddI); Viread(tenofovir disoproxil fumarate, TDF); Zerit (stavudine, d4T); Ziagen(abacavir, ABC); Amdoxovir (AMDX, DAPD); Tenofovir alafenamide fumarate,TAF.

Examples of anti-HIV drugs include the following protease inhibitors:Aptivus (tipranavir, TPV); Crixivan (indinavir, IDV); Invirase(saquinavir, SQV); Kaletra (Aluvia, lopinavir/ritonavir, LPV/r); Lexiva(Telzir, fosamprenavir, FPV); Norvir (ritonavir, RTV); Prezista(darunavir, DRV); Reyataz (atazanavir, ATV); Viracept (nelfinavir, NFV);Prezcobix (Rezolsta, darunavir/cobicistat); Atazanavir+Cobicistat.

Examples of anti-HIV drugs include the following integrase inhibitors:Isentress (raltegravir, MK-0518); Tivicay (dolutegravir, S/GSK-72);Vitekta (elvitegravir, GS-9137).

Examples of anti-HIV drugs include the following fusion inhibitors:Fuzeon (enfuvirtide, ENF, T-20); Selzentry (Celsentri, maraviroc,UK-427,857)

The above stated anti-HIV drugs are exemplary and other anti-HIV drugsare within the scope of this description.

The aTh1 compositions and/or the AVI compositions described herein canbe used in methods to reduce and or eliminate HIV from patients. Themethods described herein can enhance the CD4+ cells in the patient. Themethods can also reduce the viral load and/or purge the virus from thepatient.

CD4 Enhancement Method

The methods included in the present description can include the CD4Enhancement Method. The CD4 Enhancement Method can use the aTh1composition in a HIV patient taking anti-viral medication. This canincrease the CD4+ cell counts of HIV patients, preferably the Th1 memory(CD4+CD45RO+) cells. The new CD4+ cells created by the method can beresistant to viral replication and viral entry. The CD4 EnhancementMethod can be used in patients that have immunological failure on HAARTmedication.

The CD4 Enhancement Method can increase CD4+ cell counts by creatingactivated CD4+ Th1 memory cells in circulation. Activated CD4+ Th1memory cells can be resistant to HIV replication. This HIV resistantstate can be due to an increase in the production of chemokines that arereleased from activated memory cells which in turn interact with theCCR5 receptor (i.e., RANTES, MIP-1alpha and MIP-1beta) and due to thedown-regulation of CCR5 expression on CD4 memory cells that areactivated.

The creation of viral resistant CD4+ cells can be an important aspect ofthe CD4 Enhancement Method. Methods that would increase numbers of naïveCD4 cells, Th2 cells, Th0 cells or resting CD4 memory cells would onlyadd “fuel to the fire”. These undesirable CD4 subtypes are permissivefor viral replication. “Fuel for the fire” means that there would bemore CD4 targets for viral entry and thus more cells may be producingvirions into plasma, which can lead to more cells with latent infection,increasing viral load and eventually resulting in increased CD4 celldeath. Eventually the loss of CD4 cells would reduce the CD4 countsbelow the original baseline, causing the patient to be worse off thanprior to the therapy.

The CD4 Enhancement Method can create high titers of activated Th1memory cells that are resistant to viral entry and replication due toactivation with CD28 co-stimulation (through the co-stimulatory ligandsCD80 and CD86 ligands up-regulated on APC) that can cause an increase inexpression of native CCR5 ligands and the concomitant down regulation ofsurface CCR5 expression. To create these HIV resistant cells, the methodcan include multiple injections of the aTh1 composition (priming doses)and activation of APC to express CD80 and CD86 co-stimulatory molecules.The priming doses of the aTh1 composition can be administeredintradermally, subcutaneously, intramuscularly or intravenously. TheaTh1 composition could also be administered by a combination of theseroutes.

In one embodiment, the aTh1 composition priming doses are administeredmultiple times intradermally. A minimum of two intradermal injections ordoses can be required in order to cause memory cells to develop, such asabout 4 or more doses are administered. The doses can generally befrequent. The doses may be administered up to about 2 weeks apart, orabout 1 week apart, and even about 3-4 days apart. Doses less than about2 days apart are integrated and still considered a single dose. OnceCD4+ memory cells can be detected in the circulation, the patient can besaid to be ‘primed’ (i.e., immune to the antigen(s) in the aTh1composition).

The CD4 Enhancement Method can result in the increase in absolute CD4+cell counts. The CD4/CD8 ratio may increase or remain near the same asbaseline due to a concomitant increase in CD8 cells. In addition, themethod can result in a shift in the Th1/Th2 balance to favor Th1. HIVinfection causes a loss of Th1 cells resulting in Th2-dominated immunecells in circulation. The methods described herein can correct thisimbalance by increasing the Th1 cell component.

In one embodiment, at least two doses of the aTh1 compositions areadministrated in the same location. After at least two doses in the samelocation, a new location may be selected for administration ofsubsequent doses. Alternatively, all doses can be administered in thesame location. If a new location is selected, at least two doses shouldbe administered at each new location. This cycle of administering dosesof aTh1 composition can continue until the desired CD4+ cell count isobtained.

Intradermal doses of the aTh1 composition in the same location can beadministered to assure that professional antigen presenting cells (APC),such as Langerhans cells (LC), macrophages (M) and immature dendriticcells (DC) that traffic to the injection site are exposed to the type 1cytokines and CD40L in the aTh1 composition at the time they engulf thehighly immunogenic antigen(s). It may take 2-3 days before these APCtraffic to the administration site. After intradermal administration, LCof the skin can engulf and process the antigens from the aTh1composition resulting in the activation and priming of antigen-specificT cells.

Type 1 cytokines and CD40L in the aTh1 composition can cause theprofessional APC that process the aTh1 antigen(s) to mature and expressMHCI/II, CD80/86 and IL-12. These mature APC may then traffic to thedraining lymph nodes to interact with naïve T-cells causing theactivation, differentiation and proliferation of new effector CD4+ Th1cells and CD8+ CTL (Tc1) killer cells specific for the antigen(s) in theaTh1 composition. Multiple administrations can convert the effectorTh1/Tc1 cells to memory cells. In the presence of anti-retroviral drugs,as the administration of the number of doses of aTh1 compositionincrease eventually a new, higher CD4 set-point can be achieved. Duringthe course of the aTh1 injections, CD4 counts and viral load can bemonitored.

This CD4 Enhancement Method can result in the patient being ‘primed’ andimmune to the antigen(s) in the aTh1 composition. This can result in anincrease in memory CD4 cells that are resistant to viral entry uponactivation. Multiple priming injections of the aTh1 composition arepreferable. Such a ‘pulsed’ introduction of antigen to the immune systemcan cause an enhanced delayed-type hypersensitivity (DTH) response atthe injection site. The DTH reactions are mediated by memory Th1 cellsand the appearance of a DTH reaction at the injection site can confirmpresence of CD4 memory cells specific for the aTh1 antigen(s). IncreasedDTH skin reaction can also correlate with increased titers of CD4 memorycells in the circulation of HIV+ patients.

The CD40L and type 1 cytokines in the aTh1 composition cannon-specifically (polyclonally) activate memory T-cells. When memory Th1cells are polyclonally activated, they can expand and maintain a HIVresistant memory, CCR5− phenotype. The expansion of HIV-resistant, CD4memory cells can cause a beneficial sustained increase in CD4 counts. Inorder to polyclonally activate circulating CD4 memory cells, the aTh1composition may be infused intravenously.

Intravenous infusion of aTh1 composition may also activate latentlyinfected memory cells. These activated cells can begin to produce virusupon polyclonal activation. The method described herein can create apool of memory CD4 cells resistant to virus, these cells can providehelp for HIV-specific CTL killer cells to eliminate cells that areactively producing virus. If the patient remains on HAART medication,the viral production can be slowed so that the CD4 count can bemaintained high enough to support the anti-HIV immune response. In thismanner, the resident anti-HIV immune response can identify and kill theactivated memory cells producing virus while new viral resistant memorycells are replacing these cells. This balance between immune eliminationof activated cells producing virus and increase in viral-resistantmemory cells eventually leads to an increase in absolute CD4 counts anda decrease in the latent viral burden. Fluctuations in CD4 counts mayoccur prior to reaching the higher CD4+ cells set point level.

After a patient is primed and the CD4 count has increased, the CD4counts can be further increased and the memory cells can be continuouslyprotected from HIV elimination by the simultaneous intradermal injectionof the aTh1 composition and the intravenous infusion of the aTh1composition. The polyclonal activation of Th1 memory cells incirculation can cause the establishment of a sustained type 1 cytokinestorm. The intravenous infusion can cause activation of memory CD4 cellsin the blood of HIV patients, which in turn can cause an increase in theproduction of type 1 inflammatory cytokines, creating a type 1 cytokinestorm. Type 1 cytokines can polyclonally activate by-stander memorycells thus creating a positive feed-back loop for the maintenance ofactivated memory cells.

Activated memory cells can expand in the presence of type 1 cytokines,thus accelerating the increase in the circulating CD4 counts. A suddenand violent immune reaction is known to occur with a cytokine stormcontaining type 1 cytokines such as TNF-alpha and IFN-gamma. Such acytokine storm can be beneficial to HIV patients. Also type I cytokinessuch as IFN-gamma and IL-12 can enhance the memory cell function andinnate immune activity.

In preferred embodiments where the aTh1 composition used is AlloStim™,the intravenous infusion further enhances CD4 counts of HIV resistantmemory cells due to the CD3/CD28 coated microbeads attached to the cellsin this composition. These microbeads can also interact and activatehost memory cells causing them to proliferate. Memory cells activatedwith CD3/CD28-coated microbeads can resist HIV infection.

In one embodiment, AlloStim™ cells are used as the aTh1 composition. TheAlloStim™ cells are injected intradermally at a dose of between about0.2×10⁶ cells to about 2×10⁶ cells, preferably about 1×10⁶ cells. Anintravenous preferred dose for accelerating CD4 counts is between about1×10⁷ and about 3×10⁷ cells (low dose). AlloStim™ cells are suspended inbuffer solution (e.g., PlasmaLyteA with 1% human serum albumin) at aconcentration of about 1×10⁷ cells/ml.

One method for accelerated CD4 count enhancement can include one or morelow dose intravenous AlloStim™ infusions during the intradermal priming.The low dose intravenous infusions may occur within 7 days of the lastintradermal injection, or within 24 hours, or at the same time as anintradermal injection. The intravenous dosing does not start until atleast two intradermal priming doses have been administered, or after 4intradermal priming doses or after more than 4 intradermal primingdoses.

Variations on the timing, amounts and routes of administration can varyand all are within the scope of the present description.

Viral Load Reduction Method

The Viral Load Reduction Method can reduce viral load throughenhancement of cellular immune control of the virus. This method can beuseful in patients that are virological failures on HAART medication.The CD4 Enhancement Method and the acceleration of this method can alsoaccomplish a reduction in viral load. However, the formed methods canrequire a resident anti-HIV immune response to exist that can beawakened by the increased CD4 counts. Some patients may not have aneffective, resident anti-HIV immune response and thus are unable tomediate the immune elimination of cells that have been activated toproduce virus. In this circumstance, the Viral Load Reduction Method canbe helpful as it imprints the missing anti-HIV immune response so thatthe CD4 counts can be increased and the viral load decreased.

The Viral Load Reduction Method can include one or more HIV antigencomponents that are administered together with the aTh1 composition. TheHIV antigen components can include, for example, whole attenuated virus,as well as natural or recombinant HIV viral proteins. These HIV antigensare administered together with the aTh1 composition at the same routeand frequency of administration.

The HIV antigens and aTh1 composition are administered intradermallytogether or immediately following each other in a patient that has beenpreviously primed. The aTh1 antigens can attract a vigorous memoryresponse due to the prior priming. The viral antigens and the aTh1antigens can then be engulfed by scavenger APC, such as LC or DC. Thesecells can process and present the antigens to activate HIVantigen-specific T-cells. By this method, the aTh1 composition togetherwith the Th1 memory cells that arrive at the injection site due to theprior priming, can both act as an adjuvant to steer the development ofTh1/Tc1 anti-HIV immunity.

The Viral Load Reduction Method generally includes HIV antigens for usetogether with the aTh1 composition. These HIV antigens can be natural orrecombinant viral proteins, including tat, env and gp120. Wholeattenuated virus or virus attenuated by nef substitution can also beused. The proteins can be expressed in a carrier such as pox virus. In apreferred embodiment, the HIV viral protein is the gag protein. Therepeated administration of HIV antigens together with the aTh1composition can establish high titers of CD4 Th1 memory cells and CD8memory CTL specific for HIV. These memory cells can be maintained in anactivated state by infusing the aTh1 composition intravenously.

Viral Purge Method

The Viral Purge Method can include escalating intravenous doses of theaTh1 composition in patients on anti-viral medication. This method isused in patients that have been first subjected to the CD4 EnhancementMethod and/or the Viral Load Reduction Method. The Viral Purge Method isadministered to patients that have achieved an increased CD4 set-pointconsisting of viral-resistant memory cells. If the patient has a highlatent viral load, activation of these cells by intravenous infusion maycause a burst of viral release and may result in an immediate drop inCD4 counts. Thus it is safer to start the method from as high a CD4set-point as possible. As an example, the patient is at a CD4set-point >300 cells/ml, or at a set-point >500 cells/ml or at aset-point of >700 cells/ml.

In certain embodiments, patients that have been previously primed andhave at least a 6 month history of viral load below the limit ofdetection are subjected to increasing intravenous doses of the aTh1composition while maintaining active anti-viral suppression. Theintravenous infusions can occur at least about 3 days apart. After eachinfusion, the viral load can be assayed to determine if a viral spikehas occurred. A spike is any reading over the limit of detection. Thedoses of aTh1 can be increased at each infusion until a viral spikeoccurs. The appearance of a viral spike can be indicative that cellsfrom the latent pool have been activated. After a viral spike occurs,the CD4 counts and viral load can be followed until the viral loadreturns to the undetectable level. When the viral load is undetectable,another IV infusion at the same dose as caused the viral spike can beadministered. If again a viral spike is detected, the patient isfollowed until the viral load returns to baseline and the process can berepeated until no viral spike occurs after the intravenous infusion. Atany time no viral spike is detected, the intravenous dose can be againescalated. If the escalated dose causes a viral spike, the process isrepeated until no spike is produced. At the point that an escalatedintravenous dose does not cause a viral spike, the intravenous dosingcan be halted.

Once the intravenous doses are halted, the patient CD4 and viral loadcan be continued to be monitored. When the CD4 stabilizes with at leasttwo counts a week apart above the baseline CD4 value and the viral loadis undetectable, the patient can be taken off the anti-viral medication.While on a holiday from anti-viral medication the patient should bemonitored for CD4 count and viral load. The patient should remainwithout anti-viral drugs until the viral load spikes. When the viralload spike occurs, the anti-viral medication should be started againimmediately. After the spike of viral load on anti-viral drug holiday,the process of escalating intravenous doses can be re-initiated. Eachtime the patient is placed on holiday from anti-viral medication, thetime it takes for a viral spike to occur should be increased.

In embodiments where AlloStim™ is used as the aTh1 composition, theescalating intravenous dosing can start at about 3×10⁷ cells and canescalate to about 5×10⁷ cells to about 10×10⁷ cells to about 15×10⁷cells to about 20×10⁷ cells. Dose escalation can continue at intervalsof 5×10⁷ cells to a maximum of 100×10⁷ cells.

As discussed above, combinations of the CD4 enhancement method, theviral load reduction method and the viral purge method can beadministered. In some embodiments, the patient is administered HAART asappropriate in conjunction with the aTh1 composition.

EXAMPLES Example 1

During the initial protocol treatment phase, patients are kept on HAARTtherapy. After detecting a spike in viral burden, indicating successfulactivation of latent virus, followed by a decrease in viral burden tobaseline, indicating immune control patients can be eligible for theHAART interruption phase.

To minimize the risk of treatment interruption, patients are closelymonitored and resume treatment should virus replication be detected.

The protocol alters between intradermal and escalating intravenousdosing of AlloStim™ in patients on HAART. The intradermal dosing isdesigned to increase the titer of circulating CD4+ Th1 memory cells thatare resistant to HIV infection. The intravenous infusion is designed toprovide an inflammatory cytokine storm and activate memory CD4 cells andmacrophages (through CD40-CD40L). Activation should stimulate latentvirus replication within these reservoirs. In addition, the intravenousinfusion should activate NK cells which will target and kill viralreplicating cells providing a source of viral antigens. Dendritic cellswill process the shed viral antigens and in the inflammatory environmentstimulate anti-HIV-specific immunity. The continuous inflammatory stormwill disable viral immunoavoidance mechanisms permitting clearance ofcells with replicating virus The cycling between intradermal injectionsto increase CD4 cells and intravenous infusions to activate latent virusand stimulate anti-HIV immunity is expected to clear latent virus. EachIV infusion should cause a spike in viral load and subsequent immunecontrol should then gradually decrease viral load. If there isdifficulty in returning viral loads to baseline viral blocker drugs willbe added (such as Maraviroc and/or Fuzeon).

Regimen

The initial protocol is 28 days.

Day 0: Intradermal AlloStim™

Day 3: Intradermal AlloStim™

Day 7: Intravenous AlloStim™ (1 ml)

Day 10: Intradermal AlloStim™

Day 14: Intradermal AlloStim™

Day 17: Intravenous AlloStim™ (3 ml)

Day 21: Intradermal AlloStim™

Day 24: Intradermal AlloStim™

Day 28: Intravenous AlloStim™ (5 ml)

Viral load and CD4/CD8 ratio is measured at baseline (Day 0) and Days10, 21 and 29) and every 28-32 days thereafter for 6 months.

Research blood (45 ml) is drawn at or before baseline (Day 0) and Day 7,17, 27 before IV infusions. PBMC and plasma is stored frozen untilanalyzed for Th1/Th2 balance (ELISPOT), HIV-specific immunity (ELISPOT),cytokine bead array.

Phenotype analysis is conducted prior to baseline and Day 60 (+/−2 days)including:

CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CD25

CD14, HLA-DR, CD80, CD86, CD16, CD38, CD117

CBC, CMP, CRP laboratory tests for safety taken at baseline, Day 7, Day14, Day 21 and Day 28.

HAART Interruption

Access to lymphoid tissue or most anatomic compartments in otherwisehealthy HIV subjects in order to determine level of latent infection isdifficult. Further, even if such studies fail to detect an infectedreservoir, they cannot prove latent virus eradication. The ultimate testof efficacy can only be the withdrawal of HAART.

Patients that experience a spike in viral load and then recover tobaseline or lower and remain at base line or lower for at least 60 daysare provided the option of entering into a HAART interruption phase ofthe protocol. In this phase, all viral suppressive drugs are withdrawnand viral load will be measured daily for the first 7 days. If anincrease in viral load is detected, HAART will be re-started. If no risein viral load is detected, the HAART interruption is continued withviral load being measured weekly for 7 weeks. If no viral load increaseis detected, monthly viral burden tests are conducted until the 1 yearanniversary of the HAART interruption. At any time an increase in viralburden is detected, HAART is reinitiated.

Primary Outcome Measures:

Changes in steady state viremia (so-called viral set point) at baselineand monthly for 6 months after completion of 28 day protocol whileremaining on HAART.

Safety and tolerability

Changes from baseline and absolute counts and activation status of CD4and CD8 naïve and memory T cells

Changes in absolute counts and activation state of monocyte/macrophages

Changes in the number of interferon (IFN)-gamma generating (in responseto HIV antigens) CD4 T cells/million peripheral blood mononuclear cells(PBMCs) as measured by intracellular cytokine staining (ICS) or ELISPOT.

Secondary Outcome Measure:

Time to viral burden increase from baseline after HAART interruption

Inclusion Criteria:

HIV-1-infected

On a stable HAART regimen without changes or interruptions for at least12 weeks. prior to study entry. Patients must be currently takingregimens containing drugs of at least two different classes.

Two readings of plasma HIV-1 viral load of less than 50 copies/ml within30 days prior to study entry.

CD4 count greater than 350 cells/mm{circumflex over ( )}3 within 12weeks prior to study entry.

Lowest CD4 count greater than 250 cells/mm{circumflex over ( )}3 at anytime prior to study entry.

Willing to use acceptable forms of contraception.

Karnofsky performance score 90 or higher obtained within 30 days priorto study entry.

Exclusion Criteria:

Age <18 years old.

Patients with failure to HAART.

HIV-1 viral load greater than 500 copies/ml within the 24 weeks prior tostudy entry.

History of any chronic autoimmune disease (e.g., Graves' disease).Excessive exposure to the sun (e.g., sunbathing, tanning bed) within 2weeks prior to study entry.

Previous CDC Category B or C event.

Use of immunomodulating therapy, including cyclosporine, IgG-containingproducts, interleukins, interferons, or systemic glucocorticosteroids(including those inhaled) within 6 months prior to study entry.

Exposure to an experimental HIV vaccine.

Any vaccine within 30 days prior to study entry.

Investigational products within 12 weeks prior to study entry.

Current drug or alcohol use or dependence that, in the opinion of theinvestigator, would interfere with the study.

Serious illness requiring systemic treatment and/or hospitalization.Participants who complete therapy or are clinically stable on therapyfor at least 14 days prior to study entry are not excluded.

Positive hepatitis B surface antigen or positive anti-hepatitis Cantibody at screening.

Pregnant or breastfeeding.

Adequate organ function including:

Marrow:

WBC>3000/mm³

Platelets>100,000/mm³.

Absolute neutrophil count ≥1,500/mm³

Hemoglobin≥10.0 g/dL (transfusion allowed)

Hepatic:

Serum Total bilirubin<1.5×ULN mg/dL,

ALT (SGPT)/AST (SGOT)≤1× upper limit of normal (ULN).

Renal:

Serum creatinine (SCR)<1.0×ULN, or

Creatinine clearance (CCR)>30 mL/min.

History of cardiac, pulmonary, gastrointestinal, hepatic, renal,pancreatic, or neurologic disease which, in the opinion of the studyofficial, will compromise study participation

Example #1

A HIV+ man on HAART medication for 19 years with viral load always belowdetectable limits was entered into the Viral Enhancement protocol.

The patient had absolute CD4 cell counts of 250-350 at baseline.

He was administered 1×10⁷ AlloStim™ intradermally while on his HAARTmedication on Day 0, Day 3 in same location. Then again on Day 7 and Day10 in another location. Over this period of time, his absolute CD4 countincreased from 350 cells to 450 cells.

Beginning on day 14 escalating intravenous doses of AlloStim™ wereadministered. On day 14, 1×10⁷ cells were infused. There was nodetectable viral load. On day 17, 5×10⁷ cells were infused. There was nodetectable viral load. On Day 21, 10×10⁷ cells were infused. The viralload spiked to 66 and remained above detection for 10 days when it againreturned to undetectable. CD4 count increased to over 500 during thisperiod and continued to rise over the next 60 days stabilizing at over600.

Example #1

A HIV positive man on HAART medication for at least six years withundetectable viral load. His absolute CD4 counts ranged from 100-230over a period of 2 years.

The patient had a 250 CD4 count at baseline.

He was administered 1×10⁷ AlloStim™ intradermally on Day 0, Day 3, Day10 and Day 14. His CD4 counts increased to 293. On Day 17 he received a1×10⁷ intradermal injection and a 3×10⁷ intravenous infusion. On day 21he received a 1×10⁷ intradermal injection and a 10×10⁷ intravenousinfusion. On Day 24 he received a 10×10⁷ intravenous infusion. On Day 28and day 31 he received a 10×10⁷ intravenous infusion. His viral loadspiked at 300 on day 31 and returned to baseline by day 42. During thistime his CD4 counts slowly declined to below 200 by day 42.

Beginning on Day 49 until Day 63, he received 1×10⁷ intradermalinjection of AlloStim™ every 3-4 days. His CD4 count gradually increasedfrom below 200 to above 300. His viral remained undetectable.

On Day 84, Day 87, Day 91 and Day 94 he received 10×10⁷ intravenousAlloStim™ infusions. On Day 97 his viral load spiked to 86. By Day 101,his viral load returned to baseline and his CD4 counts remained over300. He was removed from his HAART medication. He remained withundetectable viral load for 31 days without HAART medication. On Day 32the viral load was 300 and CD4 230. HAART was restarted and the viralcame back to undetectable and CD4 stabilized at around 250.

Although the present description has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A method of treating a patient with HIVcomprising: increasing the titer of circulating CD4+ Th1 memory cellsthat are resistant to HIV infection by administering at least twointradermal doses of aTh1 composition to the patient, wherein thepatient is infected with HIV; and expanding and activating the CD4+ Th1memory cells in the patient by administering at least two intravenousdoses of the aTh1 composition, wherein the first intravenous dosecomprises the smallest amount of the aTh1 composition and eachsubsequent intravenous dose has an escalated amount of the aTh1composition than the preceding intravenous dose, wherein the patient isconcurrently treated with highly active antiretroviral therapy (HAART)and one or more HIV antigens.
 2. The method of claim 1 wherein theintradermal doses are administered at the same location.
 3. The methodof claim 2 wherein the interval between the intradermal doses is betweenabout 3 days and about a week.
 4. The method of claim 2 furthercomprising an additional two intradermal doses of the aTh1 compositionat a location different than the location of the first two intradermaldoses.
 5. The method of claim 1 wherein the at least one intravenousdose is administered within about 3 days of the last intradermal dose.6. The method of claim 1 wherein the intravenous dose is administered atabout the same time as the last intradermal dose.
 7. The method of claim1 wherein the aTh1 composition comprises allogeneic activated CD4+T-cells.
 8. The method of claim 1 wherein the latent viral load isreduced or eliminated in the patient.
 9. The method of claim 1 whereinthe expanding and activated step comprises administering at least threeintravenous doses, wherein the second dose of the intravenous aTh1composition has an escalated dose relative to the first dose of theintravenous aTh1 composition and the third dose of the intravenous aTh1composition has an escalated dose relative to the second dose of theintravenous aTh1 composition.